Contribution of TCR Signaling Strength to CD8+ T Cell Peripheral Tolerance Mechanisms

Trevor R. F. Smith, Gregory Verdeil, Kristi Marquardt and Linda A. Sherman
J Immunol October 1, 2014, 193 (7) 3409-3416; DOI: https://doi.org/10.4049/jimmunol.1401194

Abstract

Peripheral tolerance mechanisms are in place to prevent T cells from mediating aberrant immune responses directed against self and environmental Ags. Mechanisms involved in the induction of peripheral tolerance include T cell–intrinsic pathways, such as anergy or deletion, or exogenous tolerance mediated by regulatory T cells. We have previously shown that the density of peptide-MHC class I recognized by the TCR determines whether CD8+ T cells undergo anergy or deletion. Specifically, using a TCR-transgenic CD8+ T cell model, we demonstrated that persistent peripheral exposure to low- or high-dose peptides in the absence of inflammatory signals resulted in clonal deletion or anergy of the T cell, respectively. In this study, by altering the affinity of the peptide-MHC tolerogen for TCR, we have confirmed that this mechanism is dependent on the level of TCR signaling that the CD8+ T cell receives. Using altered peptide ligands (APLs) displaying high TCR affinities, we show that increasing the TCR signaling favors anergy induction. Conversely, using APLs displaying a decreased TCR affinity tilted our system in the direction of deletional tolerance. We demonstrate how differential peripheral CD8+ T cell tolerance mechanisms are controlled by both the potency and density of MHC class I–peptide tolerogen.

Introduction

The mechanism of negative selection blocks T cells displaying TCRs with high affinity for self-peptide–MHC complexes from escaping the thymus (1). However, this process is not absolute, and significant numbers of T cells that recognize and respond to peripheral self-peptide–MHC molecules emigrate from the thymus (2). Fortunately peripheral tolerance mechanisms, such as anergy, deletion, and T regulatory cells, are in place to protect against dangerous autoreactive T cell responses (3–5). The identification of the factors controlling the induction of such mechanisms is the focus of much research, as once harnessed these mechanisms can be exploited to prevent or treat a myriad of immune disorders.

In a steady-state system where T cell activation through the TCR (signal 1) occurs in the absence of costimulation (signal 2) and proinflammatory cytokines (signal 3), we and others (6, 7) have observed that the mechanism of peripheral tolerance induced was dependent on the level of Ag in the milieu. Following exposure of CD8+ T cells to a persistent low dose of Ag, they initially expand, but soon undergo apoptosis through a Bim-dependent mechanism (6). On the other hand, a high Ag dose rendered the CD8+ T cells unresponsive (anergic) to TCR signals, but cells survived. Upon removal of Ag, these cells became a pool of memory cells that could later respond to Ag (6).

The ability to induce differential tolerance mechanisms by varying the level of Ag exposure suggests the strength of TCR signaling as a controlling factor. In previous studies the density of Ag was varied, rather than the affinity of the TCR for MHC-peptide. As both Ag affinity and Ag concentration can contribute to the overall strength of TCR signal, we have evaluated whether affinity differences are also able to determine whether tolerance occurs through deletion or anergy. We hypothesized that by using altered versions of the tolerizing peptide, we could control whether we induced CD8+ T cell tolerance through deletion or anergy. Furthermore, all the initial conclusions were based on studies of a single TCR exhibited by CL4 CD8 cells. In this article, we generalize our findings by extending these studies to other TCRs expressed by CD8 cells.

Materials and Methods

Mice

B10.D2, C57/BL6, Bim−/−, and NOD mice were purchased from The Jackson Laboratory or the Animal Breeding Facility at the Scripps Research Institute (La Jolla, CA). B10.D2 CL4 and CL1 TCR transgenic mice have been described previously (8, 9). OT-1 TCR transgenic mice were provided by Dr. Charlie Suhr (The Scripps Research Institute, La Jolla, CA). All animals were bred at our facilities and housed under specific pathogen-free conditions. All animal experiments were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Scripps Research Institute.

Peptides and immunizations

Influenza HA518-526 peptides (native and variant sequences) were synthesized by GenScript (Piscataway, NJ). OVA257-264 (SIINFEKL) and A2 variant were a gift from Dr. N. Gascoigne (TSRI, La Jolla, CA). Influenza virus A/PR/8/34 H1N1 (PR8) was grown in the allantoic cavity of 10- to 11-d-old hen’s eggs. Upon isolation, the allantoic fluid was titrated for hemagglutination (HA) activity using chicken RBC and stored at −70°C. Mice were infected i.p. with 500 HA units of PR8 virus. Recombinant Listeria monocytogenes–expressing OVA was provided by Dr. C Suhr (TSRI, La Jolla, CA). Mice were infected i.v. with 1 × 105 CFU recombinant Listeria monocytogenes–expressing OVA.

Preparation and adoptive transfer of naive TCR transgenic T cells

CD8+ T cells were isolated from the lymph nodes of CL1, CL4, or OT-1 TCR mice (6–8 wk old) by negative selection using the CD8+ T cell isolation kit (BD Bioscience). T cell purity was >90% with no contaminating CD4+ cells. For adoptive transfer experiments, the indicated number of cells was injected i.v. in 100 μl HBSS.

Flow cytometry

Suspensions of spleen cells were stained for FACS analysis in HBSS containing 1% FCS and 2 mM EDTA with the following mAbs (from BD Biosciences unless otherwise stated): CD8α-PE (53–6.7), CD44-FITC (IM7), NKG2a-FITC (20d5), Thy1.1-PerCP.Cy5 (OX-7), and PD-1-APC (29F.1A12; BioLegend).

For ex vivo detection of Erk phosphorylation, splenocytes were harvested and processed to single-cell suspensions, placed in a 96-well plate at 2 × 106 cells/well, and restimulated with 1 μg/ml Kd HA peptide, or PMA (200 ng/ml) and ionomycin (12.5 μg/ml) in RPMI 1640 medium containing 10% heat-inactivated FCS for 15 min at 37°C, in a 96-well plate. Immediately following Ag restimulation, cells were fixed in fresh 2% paraformaldehyde for 10 min at room temperature and then permeabilized with ice-cold 90% methanol for 1 h at 4°C. After washing, cells were stained with Thy1.1-PerCp.Cy5 and CD8α-PE (BD Bioscience) for 30 min at 4°C. After washing, phospho-Erk1/2-APC (pT202/pY204; BD Biosciences) was added and cells stained for 1 h at room temperature. Cells were washed twice with HBSS containing 0.1% w/v BSA (Sigma-Aldrich) and 0.02% w/v sodium azide. The cells were analyzed immediately with a FACSCalibur and FlowJo software (Becton Dickinson).

Loading of spleen cells with isoleucine, leucine, alanine 3 amino acid extension to peptide

Isoleucine, leucine, alanine (ILA) 3 amino acid extension to peptide–loaded spleen cells were prepared by osmotic shock using the method described by Liu et al. (10). Briefly, 150 × 106 splenocytes from B10.D2 mice were washed twice in RPMI 1640 medium and resuspended in 1 ml of hypertonic medium (0.5 M sucrose, 10% w/v polyethylene glycol 1000, and 10 mM HEPES in RPMI 1640 [pH 7.2]), containing peptides, for 10 min at 37°C. Next, 13 ml of prewarmed hypotonic medium (40% H2O, 60% RPMI 1640) was added, and the cells were incubated for an additional 2 min at 37°C. Immediately thereafter, the cells were pelleted by centrifugation and washed twice with ice-cold HBSS, and 30 × 106 in 0.2 ml ILA-loaded splenocytes was injected i.v. into each recipient mouse as a source of dying cells.

Statistical analysis

Data are expressed as mean + SD for each group. Statistical differences between groups were evaluated with a Student t test using GraphPad Prism 5.0b software; p < 0.05 was considered statistically significant.

Results

Peripheral tolerance induction using high affinity peptides promotes CL4 CD8+ cell unresponsiveness

Previous studies performed in our laboratory used CD8+ T cells from CL4 transgenic mice, expressing a TCR that recognizes the Kd-restricted HA peptide (IYSTVASSL) derived from the influenza HA virus (8). We demonstrated that daily i.v. injections of low-dose (0.1 or 1 μg) KdHA for 3 d resulted in the deletion CL4 CD8+ T cells (6). However, daily i.v. injections of high-dose (10 or 100 μg) KdHA for 3 d induced a state of unresponsiveness (anergy) in the CL4 CD8+ T cell population (6). To determine in this study whether the mechanism of peripheral tolerance induced by cognate peptide-MHC was dependent on TCR affinity, we designed experiments incorporating altered peptide ligands (APLs) containing an amino acid substitution at a TCR contact position within the Kd-HA sequence. To test our first hypothesis, that increasing the strength of TCR signaling would promote the acquisition of an anergic phenotype, we used the Kd-HA super agonist A517G as the tolerizing peptide Ag. A517G had been shown previously to activate CL4 cells at a lower concentration (factor of 10–30) than native peptide sequence did (11). Importantly, the binding affinity of A517G for Kd was the same as the native KdHA peptide. Therefore, the substitution of a glycine for alanine at position 517 increased the TCR signaling strength directly. We found that A517G induced CL4 proliferation at ∼1 log lower concentration than native KdHA peptide (data not shown).

B10.D2 mice harboring 3 × 106 adoptively transferred CL4 cells were treated with three daily i.v. injections of a dose (1 μg) of native peptide Ag shown previously to delete all CL4 cells, or the same amount of A517G peptide. On the third day, groups of mice were sacrificed and the transferred CL4 cells analyzed. CD44 was equally upregulated on all CL4 cells in both Ag treatment groups, indicating all transferred CL4 cells had been exposed to Ag (Fig. 1A). Previous experiments in which CL4 cells undergoing anergy versus deletion were examined for expression of markers known to be associated with exhaustion revealed greater upregulation of the negative costimulatory molecules NKG2a and PD-1 on CL4 anergic cells (G. Verdeil and T.R.F. Smith, unpublished observations). Treatment with the super agonist A517G promoted the acquisition of the anergy-associated phenotype, with increased levels of NKG2a and PD-1 expressed on the CL4 cells as compared with these same cells activated with native HA peptide (Fig. 1A). T cell anergy has been associated with reduced phosphorylation of TCR-mediated signaling molecules, such as ERK and JNK protein kinases (12, 13). We previously reported that treatment of CL4 with a low dose of HA peptide that resulted in their deletion was associated with attenuated ERK signaling as compared with untreated cells, whereas treatment with a higher dose completely abrogated activation of ERK as assessed upon restimulation through the TCR (6). Similarly, we found that treatment with 1 μg A517G abrogated ERK activation upon restimulation of CL4 cells with Kd-HA (Fig. 1B). ERK signaling was still observed in CL4 cells after treatment with 1 μg of native Kd-HA; however, it was attenuated when compared with naive CL4 cells, consistent with a partial anergic phenotype prior to deletion.

FIGURE 1.
FIGURE 1.

Use of a peptide with high TCR affinity in a peripheral tolerance protocol promotes CD8+ T cell anergy. B10.D2 mice containing donor Thy1.1+ CL4 CD8+ T cells were treated daily with PBS, 1 μg native Kd-HA or A517G peptide between days 0 and 2. (A) Day 3 flow cytometric analysis of spleen-derived CL4 cells stained with Ab specific for the indicated molecules: PBS (dashed line), native Kd-HA (thin line), and A517G (thick line) treatments. (B) Day 3 ex vivo phosphorylation of Erk in CL4 cells restimulated with Kd-HA peptide (thin line) and unstimulated (dashed line). Treatment condition is indicated in the top right corner of plots, and the mean fluorescence intensity increase from unstimulated to stimulated cells is indicated in parentheses. Survival of CL4 cells in the spleen. Expressed as the absolute number (C) or percentage (D) of CL4 cells detected by FACS on day 6 compared with day 3 for each treatment condition. (E) On day 30, mice were challenge with PR8 influenza virus (i.p. 500 HA units), and donor CL4 cells were detected by FACS in the spleen 6 d later. Each graph is representative of three to five experiments using age- and sex-matched mice. In the experiments depicted in (C) and (D), there were four to six mice per treatment group.

As stated previously, low-dose Ag treatment initiates a mechanism that results in cell death, whereas high doses of Ag induced unresponsiveness. We measured CL4 cell survival after treatment with native peptide or the superagonist. Kinetic studies revealed that the size of the splenic CL4 cell pool expands during the first 3 d of treatment with a deleting dose of KdHA, before contraction begins on day 4, and by day 6 only a fraction of the CL4 population can be detected (G. Verdeil and T.R.F. Smith, unpublished observations). We therefore defined the efficiency of deletion as a percentage of CL4 cells remaining on day 6 in comparison with day 3. Treatment with 1 μg native KdHA results in efficient deletion of the CL4 cell population, whereas A517G treatment results in a significantly higher level of survival (Fig. 1C, 1D). To confirm that all the cells treated with native peptide were deleted and that memory cells had not been formed, we measured the influenza virus recall response of any residual CL4 cells 30 d later (Fig. 1E). A robust expansion to virus challenge occurred in the mice that received PBS or 1 μg A517G treatment, but this response was negligible in mice that received 1 μg native KdHA treatment. These results indicate that treatment with a tolerogen with strong TCR signaling properties favors the induction of the anergic form of peripheral tolerance over deletion.

Peripheral tolerance induction using low TCR affinity peptides promotes CL4 CD8+ T cell deletion

Our original hypothesis predicted that peptides with low TCR affinity would favor deletion. We compared tolerance induction mechanisms induced in CL4 cells treated with equal doses of native KdHA or an APL (I512V) displaying weaker TCR affinity. I512V had previously been shown to be a weak TCR agonist, despite equivalent binding to Kd-HA (11). Thus, its weak agonistic characteristic was deemed to be due to weaker TCR interactions caused by an isoleucine to valine substitution at the 512 position. We chose to determine the tolerance phenotype induced at a dose of peptide (10 μg) that was shown to cause anergy in previous experiments using native peptide (6). We first ascertained that 10 μg treatment with I512V induced activation (CD44 upregulation) of all transferred CL4 cells (Fig. 2A). In comparison with native KdHA peptide, I512V treatment upregulated NKG2a to a similar level, but PD-1 expression was lower (Fig. 2A). The ability of CL4 cells to phosphorylate ERK was completely lost after treatment with 10 μg native KdHA, but some remained, although greatly attenuated, in I512V treated cells (Fig. 2B). Analysis of cell survival showed a dramatic difference between the two peptides; 10 μg native KdHA treatment resulted in only a slight contraction in the CL4 pool between days 3 and 6 (Fig. 2C), and influenza virus recall at day 30 induced a robust response (Fig. 2D). However, 10 μg I512V treatment resulted in a severe loss of the CL4 cell pool between days 3 and 6 (Fig. 2C), and a negligible recall response to day 30 viral challenge (Fig. 2D). Thus, signaling through the TCR with a lower affinity ligand skews the mechanism of tolerance toward deletion.

FIGURE 2.
FIGURE 2.

Use of a peptide with low TCR affinity in peripheral tolerance protocol promotes CD8+ T cell deletion. B10.D2 mice containing donor Thy1.1+ CL4 CD8+ T cells were treated daily with PBS, 10 μg of native Kd-HA, or I512V peptide between days 0 and 2. (A) Day 3 flow cytometric analysis of donor CL4 cells in the spleen stained with Ab specific for the indicated molecule: PBS (dashed line), native Kd-HA (thin line), and I512V (thick line) treatments. (B) Day 3 ex vivo phosphorylation of Erk in CL4 cells restimulated with Kd-HA peptide (thin line) or unstimulated (dashed line). Treatment condition is indicated in top right corner of plots, and the mean fluorescence intensity increase from unstimulated to stimulated cells is indicated in parentheses. (C) Survival of CL4 cells in the spleen, expressed as the percentage of CL4 cells detected by FACS on day 6 compared with day 3 for each treatment condition. (D) On day 30, mice were challenge with PR8 influenza virus (I.P 500 HA units), and donor CL4 cells were detected by FACS in the spleen 6 d later. Each graph is representative of two to three experiments using age- and sex-matched mice.

Anergy induction is impaired in CD8+ T cells expressing a low-affinity TCR

The previous experiments suggest that anergy of peripheral CD8+ T cells is induced only by potent signaling through the TCR. Therefore, if a T cell expressed a TCR displaying weak affinity for self-antigen, one would expect a deletional tolerance mechanism to be favored upon Ag recognition in the absence of signals 2 and 3. We tested this hypothesis in a system containing T cells expressing a TCR that recognized the native KdHA peptide with low affinity—CL1 (9). CL1 was originally derived from a mouse expressing HA as a self-antigen; therefore, it represents an example of a T cell that escapes thymic and peripheral tolerance because of its low affinity for self-antigen. When compared with CL4 cells, CL1 cells displayed weak Kd-HA tetramer binding, and a 10–100-fold increased concentration of Kd-HA peptide was needed to induce comparable levels of proliferation (14). To determine the susceptibility of CL1 to peripheral tolerance mechanisms, experiments were designed in which CL1 cells were subjected to the peptide tolerance treatment protocol with escalating doses (1, 10, and 100 μg) of native Kd-HA peptide, and peripheral tolerance parameters were measured. We first confirmed that CD44 upregulation was induced on transferred CL1 cells after the third daily injection of native Kd-HA, although its expression on cells in the group receiving 1 μg peptide was more variable than for the other conditions (Fig. 3A). NKG2a expression was not significantly upregulated by any dose of native Kd-HA, but the level of PD-1 increased with escalating dose (Fig. 3A). The ability to phosphorylate ERK upon restimulation through the TCR decreased with an increase of treatment dosage (Fig. 3B). However CL1 cells still displayed a higher level of ERK phosphorylation upon restimulation (Fig. 3B) than did day 3 CL4 cells treated with 10-μg doses of Kd-HA peptide (Fig. 2B). Next, we analyzed the survival of CL1 cells. By day 6, the majority of CL1 were deleted in all treatment groups (Fig. 3C); however, significant differences were observed between groups. The incomplete deletion associated with the 1-μg treatment group could be due to the presence of a significant number of cells that had not received sufficient exposure to Ag to induce deletion, as suggested by the varying levels of expression of CD44 on the CL1 cells in the 1-μg treatment group (Fig. 3A). The slight increase in CL1 cell survival in the 100-μg group compared with the 10-μg group might indicate a small proportion of the cells attained an anergic phenotype by the high concentration of peptide. However, analysis of the day 30 viral recall response failed to reveal a robust response in any treatment group (Fig. 3D). Thus, T cells expressing low-affinity TCRs are significantly more susceptible to deletion than anergy.

FIGURE 3.
FIGURE 3.

CD8+ T cells expressing a low-affinity TCR for tolerizing Ag are not anergized efficiently. B10.D2 mice containing donor Thy1.1+ CL1 CD8+ T cells were treated daily with PBS or 1, 10, or 100 μg native Kd-HA peptide between days 0 and 2. (A) Day 3 flow cytometric analysis of donor CL1 cells in the spleen stained with Ab specific for indicated molecule: PBS (shaded area), 1-μg (dashed line), 10-μg (thin line), and 100-μg (thick line) treatments. (B) Day 3 ex vivo phosphorylation of Erk in CL1 cells restimulated with Kd-HA peptide (thin line) or unstimulated (dashed line). Treatment condition is indicated in the top right corner of plots, and the mean fluorescence intensity increase from unstimulated to stimulated cells is in parentheses. (C) Survival of CL1 cells in the spleen, expressed as the percentage of CL1 cells detected by FACS on day 6 compared with day 3 for each treatment condition. (D) On day 30, mice were challenge with PR8 influenza virus (i.p. 500 HA units), and donor CL1 cells were detected by FACS in the spleen 6 d later. Each graph is representative of three experiments using age- and sex-matched mice. In the experiments depicted in (C) and (D) there were three to six mice per treatment group.

Differential tolerance induction in OVA-reactive CD8+ T cells is also dependent on dose of Ag and strength of signaling

The experiments demonstrating that differential tolerance mechanisms can be induced by alterations in dose and TCR affinity have been limited to Kd-HA–reactive CD8+ T cells on the B10.D2 background. To assess the generality of these findings, we designed experiments using a different antigenic system. C57BL/6 OT-1 CD8+ T cells recognize the OVA-derived peptide SIINFEKL in the context of Kb. Using OT-1 CD8+ T cells, we first analyzed the induction of tolerance mechanisms after a multiple-dose treatment protocol and the effect of altering the dose of tolerizing peptide (SIINFEKL). We studied the effect of tolerizing doses of 0.01, 0.1, 1, and 10 μg SIINFEKL on the induction of anergy or deletion mechanisms. Daily exposure to 1- or 10-μg doses favored the induction of anergy, and we observed upregulation of NKG2a and PD-1 (Fig. 4A) and failure to phosphorylate ERK after restimulation through the TCR (Fig. 4B). Furthermore, there was an increase in survival of OT-1 cells between days 3 and 6, compared with 0.01- or 0.1-μg treatments (Fig. 4C). Treatment with a 0.01- or 0.1-μg dose caused the efficient deletion of the OT-1 cells (Fig. 4C). Therefore, as found with CL4 CD8+ T cells, OT-1 cells are differentially tolerized depending on the dose of tolerogen. However, the phosphorylation of ERK in response to TCR stimulation on day 3 was not strictly associated with deletion. ERK activation was turned off in 0.1 μg-treated OT-1 cells undergoing deletion, but it was present in 0.01 μg treated OT-1 cells.

FIGURE 4.
FIGURE 4.

The type of peripheral tolerance induced in OVA-reactive CD8+ T cells is dependent on the dose of peptide. C57BL/6 mice containing donor Thy1.1+ OT-1 CD8+ T cells were treated daily with PBS or 0.01, 0.1, 1, or 10 μg native SIINFEKL peptide between days 0 and 2. (A) Day 3 flow cytometric analysis of donor OT-1 cells in the spleen stained with Ab specific for indicated molecule: PBS (shaded area), 0.1-μg (dashed line), 1-μg (thin line), and 10-μg (thick line) treatments. (B) Day 3 ex vivo phosphorylation of Erk in OT-1 cells restimulated with SIINFEKL peptide (thin line) or unstimulated (dashed line). Treatment condition is indicated in the top right corner of plots, and the mean fluorescence intensity increase from unstimulated to stimulated cells is in parentheses. (C) Survival of OT-1 cells in the spleen, expressed as the percentage of OT-1 cells detected by FACS on day 6 compared with day 3 for each treatment condition. Each graph is representative of two to three independent experiments using age- and sex- matched mice.

We proceeded to determine whether the induction of anergy or deletion was dependent on the agonistic strength displayed by the tolerizing Ag using the A2 altered variant of SIINFEKL. Of interest, A2 (SAINFEKL) has been described to bind to Kb equally and to possess similar TCR affinity, but displays a 10-fold lower agonist ability than the native SIINFEKL (15, 16). It has been proposed that the weaker agonistic potency of A2 is due to its weaker ability to promote interactions between CD8β and CD3ζ within the immunologic synapse (17). Because APL A2 possesses 10% of the potency of native SIINFEKL, we hypothesized that treatment with 1 μg of A2 would favor the induction of deletion. Fig. 5A shows equal expression of CD44, but failure to upregulate NKG2a and lower levels of PD-1 on OT-1 cells treated with APL A2 compared with native SIINFEKL. Day 3 after TCR stimulation phosphorylation of ERK was turned off by both APL and native peptide treatments (Fig. 5B). Analysis of OT-1 survival at day 6 and 30 d after List-OVA challenge revealed efficient deletion of OT-1 cells by 1 μg APL A2, but not 1 μg native SIINFEKL treatment protocol (Fig. 5C, 5D). In conclusion, treatment with a tolerogen that induces a weaker TCR signal, in this case because of inefficient CD8-TCR interaction, favors the induction of deletion.

FIGURE 5.
FIGURE 5.

Tolerance-inducing treatment using an APL displaying weaker agonist properties than the native peptide favors the induction of OT-1 CD8+ T cell deletion. C57BL/6 mice containing donor Thy1.1+ OT-1 CD8+ T cells were treated daily with PBS, 1 μg native SIINFEKL, or 1 μg A2 variant peptide between days 0 and 2. (A) Day 3 flow cytometric analysis of donor OT-1 cells in the spleen stained with Ab specific for indicated molecule: PBS (shaded area), 1 μg SIINFEKL (thin line), and 1 μg A2 (thick line) treatments. (B) Day 3 ex vivo phosphorylation of Erk in OT-1 cells restimulated with SIINFEKL peptide (thin line) or unstimulated (dashed line) is shown, and the mean fluorescence intensity increase from unstimulated to stimulated cells is in parentheses. (C) Survival of OT-1 cells in the spleen, expressed as the percentage of OT-1 cells detected by FACS on day 6 compared with day 3 for each treatment condition. (D) On day 30, mice were challenge with recombinant Listeria-OVA (i.v. 1 × 105 CFU). Five days later, donor OT-1 cells were detected by FACS in the spleen. Each graph is representative of two to three independent experiments using age- and sex-matched mice.

Deletional tolerance induced after activation by cross-presented Ag is dose-dependent

We have previously described an efficient method to introduce nominal CD8+ T cell epitopes into the cross-presentation pathway (18). This method is based on in vitro pulsing of spleen cells that are undergoing apoptosis with a three amino acid (ILA) N-terminal extended version of the nominal Kd-HA peptide. Upon injection, these dying cells are captured by dendritic cells that cross-present associated Kd-HA peptide to CL4 T cells. We used this model to determine whether different levels of cross-presented peptide controlled the form of peripheral tolerance induced. We treated animals with multiple doses of apoptotic cells loaded with 1 or 10 μg ILA–native HA or PBS. Upon day 30 virus recall, we observed that low-level (1 μg) cross-presentation of apoptotic cell-derived peptide led to efficient deletion (Fig. 6A), whereas cross-presentation of a high level of peptide (10 μg) failed to delete CL4 cells efficiently. We had previously shown deletional tolerance induced by peptide delivery in InsHA transgenic mice to be Bim-dependent (19). In this study, we confirmed that deletion of CL4 cells associated with a low dose of cross-presented HA Ag was also Bim-dependent, as Bim-deficient CL4 cells were not deleted (Fig. 6A).

FIGURE 6.
FIGURE 6.

Efficient deletion after constant exposure to cross-presented Ag is dependent on Bim expression, and the dose and TCR affinity of Ag. B10.D2 mice containing donor WT or Bim−/− Thy1.1+ CL4 CD8+ T cells were treated on days 0, 3, and 6 with apoptotic cells carrying various concentrations of (A) ILA–native HA or (B) ILA-A517G peptide. On day 30, mice were challenged with PR8 influenza virus (i.p. 500 HA units). Six days later, donor CL4 cells were detected by FACS in the spleen. Each graph is representative of two experiments using age- and sex-matched mice.

To determine whether deletion associated with a low dose of cross-presented peptide was dependent on the affinity of TCR signaling, we designed experiments using the ILA N-terminal extension associated with the super agonist variant A517G or native peptide. Fig. 6B demonstrates that a low dose (1 μg) of ILA-A517G does not induce efficient deletion of CL4 cells, whereas ILA-HA (native peptide) is highly efficient at deletion. Thus, the deletion of CD8+ T cells upon exposure to cross-presented Ag is dependent on the level of TCR signaling.

Discussion

Previous studies in our laboratory using CL4 CD8+ T cells revealed that the mechanism of peripheral tolerance, either deletion or anergy, was dependent on the dose of peptide and duration of exposure (3, 6). Induction of Bim-dependent deletion occurred after persistent exposure to low-dose Ag, whereas anergy was induced upon persistent exposure to a high dose of Ag. We aimed to determine whether TCR signal strength, as determined by TCR affinity rather than dose of Ag, also controlled the path of tolerance chosen by the CD8+ T cells. We also sought to determine the generality of these findings by extending them to CD8 cells expressing different TCRs.

Several different methods were selected for altering the strength of TCR signal. First we used altered peptide ligands that possessed higher (A517G) or lower affinity (I512V) than the native KdHA peptide for CL4 TCR–expressing cells did. These results indicated that the strength of signal, in this case owing to altered TCR affinity rather than density of peptide Ag, also determined whether tolerance occurred through deletion or anergy. Whereas the administration of 1 μg of native peptide resulted in deletion and 10 μg caused anergy of CL4 cell, as little as 1 μg of the superagonist was sufficient to induce anergy; 10 μg of the low-affinity agonist still resulted in deletion. We demonstrated this result in the context of absolute cell numbers and percentage change in CL4 cells between days 3 and 6. Furthermore, we showed upon systemic virus recall challenge at day 30 that there was no CL4 cell expansion, ruling out the possibility of reservoirs of undeleted cells residing in peripheral tissues. Thus, both the density of Ag and TCR affinity for MHC-peptide contribute to the mechanism of tolerance.

Next, we used the CL1 TCR that has inherently low affinity for its ligand. This HA-specific TCR has the identical specificity as CL4, except it was obtained from a mouse that expressed HA as a self-antigen and required >30-fold more Ag than CL4 to exhibit a comparable response (14). As assessed by expression of CD44, 1 μg HA peptide was sufficient to obtain stimulation of the cells in vivo; however, even when we used 100-fold more peptide, we still could not achieve anergy. The cells appeared to be obligatorily deleted. This finding suggests an affinity threshold that must be crossed before the cells can become anergic. CL1 was obtained from a TCR transgenic mouse that expressed HA in the pancreatic islets, but also express low levels in the thymus (20). It is tempting to speculate that the reason CL1 cannot become anergic is because this threshold is higher than what is permitted to escape thymic deletion in vivo. Obligatory deletion may be a safer route for the host than anergy if cells encounter a higher concentration of a self-antigen in the periphery. It is also of interest that even the highest amount of peptide used in vivo was insufficient to induce expression of NKG2A by CL1 cells, whereas there was an increase in expression of PD-1 with increasing amount of peptide, suggesting the strength of signal required for NKG2A expression is greater than for PD-1.

We also extended these studies to OT-1 cells for which many altered peptide ligands have been defined (17, 21). We were particularly interested in the A2 peptide as it binds to Kb as efficiently as SIINFEKL, and the TCR on OT-1 cells binds as well to A2-Kb as to SIINFEKL-Kb. Yet A2 is a 5–10-fold weaker agonist than SIINFEKL because of the weaker interaction between CD8 and the CD3ζ chain when binding the A2-Kb complex (17). CD8 is critical to responsiveness both through stabilization of TCR-pMHC interaction and by assisting in the delivery of Lck to the TCR-CD3 complex (22). Our results indicate that the weak agonist activity of A2 peptide extends to signaling for tolerance, as OT-1 cells were deleted rather than anergized by A2. It is interesting that nondepleting anti-CD8 Abs have been known to have beneficial effects in transplantation tolerance, yet without a full understanding of the mechanism (23, 24). Promoting deletion of allospecific CD8 cells by reducing TCR signal transduction may be one such mechanism.

We sought to determine whether cross-presented Ag was also able to induce anergy and deletion. We previously reported a method that allowed us to alter the concentration of cross-presented Ag in vivo (18). This method relied on N-terminal extension of cognate peptide by the three residues, ILA; this was sufficient to prevent recognition of peptide in vivo unless it was first processed by proteasomes in Ag presenting cells (25). This method was used to compare the fate of CL4 cells exposed to the same concentration of either native HA or superagonist, A517G. Indeed, the cells were deleted in response to ILA-HA yet underwent anergy in response to ILA-A517G. Thus, the Ag delivered to CD8 cells by professional presenting cells in vivo follows these same rules of tolerance.

Our observations suggest that the potency and the density of the ligand are interchangeable factors in determining the induction of peripheral tolerance mechanisms, so long as a threshold of TCR affinity is reached. These findings are in line with a report concerning CD4+ T cell tolerance induction by Gottschalk et al. (26). They found that weak TCR signaling associated with either low-affinity or low-density ligands resulted in comparable induction of Foxp3. However, a recent study by the same group observed distinctive molecular outcomes after an equal cumulative TCR signal is reached by ligand density in comparison with ligand potency (27). Using CD4+ TCR transgenic cells, they reported distinct influences of peptide ligand quantity when compared with ligand quality at the level of regulation of the IL-2 pathway after TCR activation, but not in the induction of proliferation. Gene expression analysis by the authors suggested that the responses mediated by TCR signals are segregated into those that are sensitive to the potency of the TCR ligand independent of ligand density, and those that are controlled by ligand density and cumulative levels of TCR signals. Although outside the scope of this study, it would be of interest to determine whether there are distinct gene expression profiles associated with the anergy phenotype after a TCR signaling threshold is reached by increased ligand density as compared with ligand potency.

In conclusion, our results suggest that, under tolerizing conditions, anergy and deletion of CD8+ T cells can be manipulated by simply altering the strength of signal delivered through the TCR. Both changes in the density of MHC-peptide or affinity of the TCR can deliver the signals required for each of these tolerance mechanisms, although there appears to be threshold of affinity that must be reached in order for anergy to occur. These results should prove of value in devising methods that are most effective in eliminating self-reactive T cells.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Dr. Nicolas Gascoigne for the OT-1 altered peptide ligands, Dr. Charles Surh for the OT-1 Tg mice, and Jocelyn Chang for mouse breeding and technical assistance.

Footnotes

  • This work was supported by National Institutes of Health Grant R0I DK050824 (to L.A.S.).

  • Abbreviations used in this article:

    APL
    altered peptide ligand
    HA
    hemagglutination
    ILA
    isoleucine, leucine, alanine.

  • Received May 12, 2014.
  • Accepted July 28, 2014.

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